Fluorescence has become one of the most important detection signals in biotechnology due to its high sensitivity and safety of handling. Processes like fluorescence resonance energy transfer (FRET) or fluorescence polarization make possible the real time analysis of biomolecular binding events, movements or conformational changes. The ability to selectively modify proteins with fluorescent probes has greatly facilitated both in vitro and in vivo studies of protein structure and function (Hermanson (1996) in Bioconjugate Techniques, Academic Press: San Diego; and Tsien (1998) Annu. Rev. Biochem., 67:509-544).
Current fluorescent methodology to study proteins in vivo often relies on fusion constructs with large fluorescent proteins such as green fluorescent protein (GFP). Although this probe has proven useful in studies of protein expression, localization and bimolecular interactions, its large size can result in significant structural perturbations. GFP fusions are also limited to the C- or N-terminus of the target protein and are relatively insensitive to their environment (Tsien (1998) Annu. Rev. Biochem., 67:509-44). GFP also requires many transcripts to achieve a suitable signal, and requires a lag-time for its folding and fluorophore maturation.
Chemical methods also can be used to selectively modify proteins with a variety of small, synthetic fluorophores that minimize structural perturbation (Hermanson, in Bioconjugate Techniques, Academic Press: San Diego (1996); Martin et al., Nat. Biotech., 23:1308-1314 (2005); Keppler et al., Nat. Biotech., 21:86-89 (2003); Lin et al., J. Am. Chem. Sci., 128:4542-3 (2006)). However, these techniques are generally limited to uniquely reactive surface accessible residues on isolated proteins (e.g., the modification of cysteine with maleimide derivatives; see Hermanson in Bioconjugate Techniques (1996) Academic Press: San Diego), exhibit poor regioselectivity, are cytotoxic or demand introduction of dye binding protein motifs.
Biosynthetic labeling methods using chemically misacylated tRNAs (Mendel et al., Annu. Rev. Biophys. Biomol. Struct. (1995) 24:435-462; Hohsaka et al., FEBS Lett. (2004) 560:173-177) have been demonstrated experimentally, but afford limited yields of protein and are typically carried out only in vitro.
The incorporation of genetically encoded fluorescent amino acids at defined sites in proteins directly in living organisms would overcome many of the limitations in fluorescence labeling of proteins (Wang and Schultz, Angew. Chem. Int. Ed. (2005) 44:34-66; Wang et al., Annu. Rev. Biophys. Biomol. Struct., (2006) 35:225-249). The site-specific incorporation of fluorescent amino acids would introduce minimum perturbation to the host protein and permit the measurement of fluorescence resonance energy transfer (FRET) with much greater precision (Truong and Ikura, Curr. Opin. Struct. Bio. 2001, 11:573-578). In addition, the use of a fluorescent amino acid will permit the probing of the local environment of each amino acid position, and pinpoint residues that mediate interaction with other cellular components by varying the position of the fluorescent amino acid in the protein. This would also be very useful to study protein folding (Lakowicz, Principles of Fluorescence Spectroscopy Ed. 2; Kluwer Academic/Plenum Publishers: New York, 1999), especially in a single-molecular system (Lipman et al., Science (2003) 301:1233-1235), because one protein molecule normally contains more than one tryptophan residue, and specific chemical labeling of proteins with fluorescent probes is problematic.
What are needed in the art are new strategies for incorporation of fluorescent unnatural amino acids into proteins for the purpose of studying protein structure and function. A general methodology has been developed for the in vivo site-specific incorporation of diverse unnatural amino acids into proteins in both prokaryotic and eukaryotic organisms. These methods rely on orthogonal protein translation components that recognize a suitable selector codon to insert a desired unnatural amino acid at a defined position during polypeptide translation in vivo. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon, and where a corresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS) charges the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the endogenous tRNAs, RSs, amino acids or codons in the host organism (i.e., it must be orthogonal). The use of such orthogonal tRNA-RS pairs has made it possible to genetically encode a large number of structurally diverse unnatural amino acids.
The practice of using orthogonal translation systems that are suitable for making proteins that comprise one or more unnatural amino acid is generally known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.” Each of these applications is incorporated herein by reference in its entirety. For additional discussion of orthogonal translation systems that incorporate unnatural amino acids, and methods for their production and use, see also, Wang and Schultz, “Expanding the Genetic Code,” Chem. Commun. (Camb.) 1:1-11 (2002); Wang and Schultz “Expanding the Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and Schultz, “An Expanding Genetic Code,” Methods 36(3):227-238 (2005); Xie and Schultz, “Adding Amino Acids to the Genetic Repertoire,” Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al., “Expanding the Genetic Code,” Annu. Rev. Biophys. Biomol. Struct., 35:225-249 (2006); and Xie and Schultz, “A chemical toolkit for proteins—an expanded genetic code,” Nat. Rev. Mol. Cell Biol., 7(10):775-782 (2006), the contents of which are each incorporated by reference in their entirety.
There is a need in the art for the development of orthogonal translation components that incorporate fluorescent unnatural amino acids into proteins, where the fluorescent unnatural amino acids can be incorporated at defined positions. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.